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Nucleic Acids Research, Vol. 18, No. 3

B to Z transitions of the short DNA hairpins formed from the oligomer sequences: d[(CG)3X4(CG)3] (X = A, T, G, C) Mohan Amaratunga, Petr Pancoska', Teodoro M.Paner and Albert S.Benight* Department of Chemistry, Box 4348, University of Illinois at Chicago, Chicago, IL 60680, USA and Faculty of Mathematics and Physics, Department of Chemical Physics, Charles University, 121 16 Prague, Czechoslovakia Received October 2, 1989; Revised and Accepted December 18, 1989

ABSTRACT Circular Dichroism (CD) spectra were collected as a function of sodium perchlorate concentration [NaCIO4] for the set of DNA hairpins formed from the oligomer sequences d[(CG)3X4(CG)3] where X = A, T, G or C. Over the range in salt concentration from 0 to 4.0 M NaCI04, the CD spectra invert in a manner characteristic of the B to Z transition. A factor analysis routine is described and employed to determine the least number of basis spectra required to fit the measured spectra of each hairpin over the entire salt range examined. In every case, linear combinations of only two sub-spectra fit the experimental spectra of the hairpins with greater than 98% accuracy, indicating the spectrally monitored structural transitions are twostate. From the relative weights of the individual subspectra, B-Z transition curves are constructed. The transitions are analyzed in terms of a simple two-state equilibrium model which yields an evaluation of the transition free-energy, AGsz, as a function of NaCI04 concentration. At 1.0 M NaCI04 and 21°C, AGBz = 5.4, 4.9, 3.6 and 2.3 kcal/mole for the G4, T4, A4 and C4 loop hairpins, respectively. INTRODUCTION Cruciform structures in DNA comprised of intramolecular stemloops or hairpins are widely believed to be crucial to genetic processes. In support of this notion, partially self-complementary DNA sequences that are able to form intramolecular cruciforms have been found near sites involved in transcription and recombination (1-5). Consequently, a number of studies investigating the physical properties of DNA hairpins have been reported (6-12). Depending on the sequence environment, transitions of local regions of supercoiled DNA to either cruciforms or left-handed Z-DNA, are two means of relieving torsional stress induced by supercoiling (13,14). Since both Z-DNA and hairpins are thought to serve as regulatory structural signals, elucidation of the physical characteristics of both secondary structures is required to understand structural switching in DNA. *

To whom correspondence should be addressed

Recently, the first crystal structure of a DNA hairpin, formed from the partially self-complementary 16 base DNA oligomer sequence d[(CG)3T4(CG)3], indicated the duplex stem region resides in the left-handed Z conformation (15). Solution NMR, CD and optical melting studies of this 16-mer (6) and several other DNA hairpins formed from similar partially selfcomplementary sequences (7-12) also demonstrated the duplex stems of these molecules adopt the Z conformation under highsalt conditions. With a single exception (10), most of these studies have focused exclusively on hairpins having single strand loops comprised of four to five thymine residues. The discovery of DNA hairpins with Z-type duplex stems establishes a model system with which to simultaneously investigate potential interactions between both types of DNA secondary structure. Although effects of loop sequence might be suspected to influence the B-Z transition of a hairpin stem, to our knowledge investigations aimed at verifying this suspicion have not been undertaken. In the study reported in this paper CD spectra as a function of NaCl04 concentration of the DNA hairpins formed from the partially self-complementary oligomers, d[(CG)3X4(CG)3] where X = A, T, G, C were measured. Over the range in salt concentration from 0 to 4.0 M NaCl04, the spectra of the hairpins invert in characteristic spectral regions indicative of the right to left-handed B-Z transition. A factor analysis routine is employed to determine the least number of basis spectra required to fit the measured spectra as a function of salt (NaCl04). Therefore, unlike earlier studies where the changes in ellipticity values from the CD spectrum at a single wavelength (6,9,16) or at most two wavelengths (7,10,17) were used to quantify the B-Z transition, all of the collected CD spectral data are utilized to construct transition curves. The analysis reveals the B-Z transitions of the hairpins are two-state and significantly loop sequence dependent.

MATERIALS AND METHODS DNA Sample Preparation DNA oligomers for this study are listed in Table I and were synthesized on an Applied Biosystems 380B automated DNA synthesizer ((3-cyanoethyl phosphoramidite chemistry) (18).

578 Nucleic Acids Research Following detritylation and deprotection, DNA samples were dried under vacuum and resuspended in ddH2O. DNAs were purified electrophoretically from 20% polyacrylamide gels by brief shadowing with short wavelength uv light (X = 254 nm) and slicing the major synthetic product out of the gel. Excised gel slices were crushed and soaked in 10 volumes of elution buffer (100 mM NaCl, 10 mM sodium phosphate, pH = 7.0) and incubated at 55°C for at least 18 hours. Gel particles were removed by filtration through 0.4 tim filters and a column of hydroxylapatite. Purified samples were exhaustively dialyzed, evaporated to dryness and stored at -20°C Post-purification electrophoresis verified fidelity of DNA samples. Prior to CD measurements, an aliquot of each DNA oligomer was dissolved in -5 ml of buffer solution (10 mM sodium phosphate, pH=6.2). DNA concentrations were determined from the extinction coefficients evaluated for each oligomer. Values of the extinction coefficients used for the hairpins in this study were 8016, 8606, 9041 and 8320 M-l cm-l for the C4, G4, A4 and T4 loop hairpins, respectively. The method employed for determining the hairpin extinction coefficients was a modification of that recently developed (19). The salt concentration of DNA solutions was adjusted by adding the appropriate amount of solid NaClO4 (twice re-crystallized, Sigma). At each salt concentration, corrections of the DNA concentration were made to account for any volume changes due to added salt. DNA concentrations were between 30 and 50 ,iM strands (-4 OD/ml). Aliquots taken from these concentrated DNA solutions, in either high or low salt, electrophoresed on 20% polyacrylamide gels revealed sharp, single bands at approximately the same migration distance as the previously characterized T4 loop hairpin (6). Likewise, the absorbance spectra above 300 nm of these DNAs as a function of salt did not indicate appreciable aggregation that might be expected to arise from intermolecular pairing, particularly for the G4 loop hairpins.

Circular Dichroism (CD) Measurements CD spectra of DNA solutions were recorded on a J-600 spectropolarimeter at ambient temperature of the sample holder (-21°C). Spectra were measured at 0, 0.1 M, 0.5 M and in increments of 0.5 M thereafter up to 4.0 M NaClO4, in a 1 cm path length quartz cell. DNA solutions were allowed to equilibrate for at least 4 hours at each titration point at 21 °C prior to recording of CD spectra. Three scans at 50 nm/min were made over the wavelength range from 185 to 330 nm. The average spectrum of these three scans was used in the analysis. Measurements were made at 10 salt concentrations. CD spectra collected at 0.1 M and 4.0 M NaCl04 on a second set of independently prepared samples were in exact agreement with those data measured on the actual sample set. From this observation the spectral data are considered to be highly reproducible.

FACTOR ANALYSIS Theory To probe characteristics of the right to left-handed B-Z transition in DNA hairpins the collected CD data of the DNAs in Figure 1 were subjected to factor analysis. Numerous versions and variations of this classical technique, applied to analyze CD spectral data from DNA and proteins, have been reported (20-25). Here we provide only a brief summary of the essential features underlying our quantum mechanical approach.

40

30

20

w)

10

0

-10

-20

185 205 225 245 265 285 305 325 Wavelength (nm) Figure 1: Circular Dichroism spectra of the DNA hairpin formed from d[(CG)3T4(CG)3] as a function of NaCIO4 concentration from 0 to 4.0 M in increments of 0.5 M at 21 'C. Units of AE are M - I -cm- 1. Analogous plots were obtained for the other hairpins as a function of salt concentration. Values of AE every 0.2 nm from 185 to 330 nm were subjected to factor analysis as described in the text.

The starting point is assuming that each collected experimental CD spectrum within a given data set, 0i(X), can be expressed as a linear combination of p basis spectra or sub-spectra, S(X), i.e.

OA(X)

a'ij Sj(w)

=

(1)

cxij are weighting coefficients of each sub-spectra (unrestricted

in sign). Because the actual input data is comprised of a number of CD spectra of the same molecule measured at different salt concentrations, equation (1) represents the columns of the input matrix, [8(X)],

[I0]

=

[SX][a]

(2)

[S(X)] is the matrix of sub-spectra with columns Sj(X). [a] is the coefficient matrix with elements, aij, the coefficient of the jt sub-spectrum in the ilh spectrum. The set of n experimental spectra, Oi(X) (i= 1,n), are considered to be continuous real functions of the wavelength, X, and thus define a finite p-dimensional Hilbert space, Hp (p c n). Equation (1) represents the expression of any experimental spectrum as a vector Oi(X) in the basis of Hp, which can be decomposed into the orthogonal subspaces generated by the basis vectors Sj(X). To find p and Sj(X), a projection operator P that projects [0(X)] onto the orthogonal subspaces of Hp must be found.

Nucleic Acids Research 579 Such an operator can be found by considering the square matrix of the input spectra defined as,

[C]

=

[O]T[O]

(3)

The elements of [C] are given by the overlap integrals

Ci= 0i(X) Oj(X) dX

(4)

In the space Hp, C is an operator with eigenfunctions Sj(X). The projection operator P that projects an arbitrary vector from Hp onto the basis formed by the eigenfunctions of C, obeys the operator eigenvalue equation, CP = P[A] (5)

[A] is the diagonal eigenvalue matrix of eigenvalues, Ai. Expressing the respective operators in matrix notation gives the matrix eigenvalue equation,

[P][C]

=

[A][P]

(6)

[P] is obtained by constructing [C] from the input data and solving the resulting eigenvalue problem in equation (6). The sub-spectra are then given by

[S(X)]

=

[O][P]

(7)

Substitution of equation (7) in equation (2) yields the coefficients, [